Candidate Genes Screening For Seed Oil Content And The Function Study Of BnaFAX1 In Brassica Napus

Rapeseed?Brassica napus,B.napus?is one of the most widely grown oil crops in the world and is an important source of edible vegetable oil in China.As compared to Canada and Europe?45⁵0%?,the oil content of rapeseed is about 42%in the main producing area of the Yangtze River Basin of China.And increasing one percentage point of the seed oil content per unit area equals to increasing the seed yield per unit area about 2.5 percentage points.Therefore,it is of great significance to explore ways to increase the oil production of Brassica napus?B.napus?.In this study,QTL and GWAS analysis for seed oil content were conducted and combined with transcriptome sequencing of extremely high-and low-oil content accessions to identify SOC-related candidate genes.Meanwhile,Since FAX1 is reported to be involved in fatty acid transport in Arabidopsis and contributes to increased biomass yield and seed oil content.Therefore,to explore whether FAX1 has the same function in Brassica napus,BnaFAX1-1 and BnaFAX1-2 were finally screened as major function genes related seed oil content by analyzing the fatty acid export?FAX?family genes in Brassica napus,and the phenotypic and functional verification analysis of heterologous overexpression in Arabidopsis thaliana and homologous overexpression in Brassica napus was used to elucidate the promotion mechanism of BnaFAX1 in oil accumulation and biomass in Brassica napus.The main research contents and results of this thesis are as follows:1.QTL mapping for seed oil contents in RIL populationIn this study,yellow seed rapeseed GH06 was used as the female parent?oil content:about 44.57%?,black seed rapeseed ZY821 was used as the male parent?oil content:about 36.69%?,and the two parental hybrid F₂ generations continued self-crossing through“one grain transfer method”.In the 10th generation,a high-generation recombinant inbred line population?RIL?containing 186 strains was constructed and cultivated in the rapeseed planting base of Xiema Town,Beibei District,Chongqing for three consecutive years?2016²018??29°45?39.99"N,106°22?38.47"E,238.57 m?.And a high-density genetic linkage map consisting of8575 SNP markers and 1201 clusters covering the genome 6140.2cM of Brassica napus was constructed by using 60K SNP chip.The reference map for QTL analysis included 5560 SNP loci,and the average distance between markers was 1.10cM.The QTLs for SOC was detected using composite interval mapping?CIM?by Win QTL Cartographer 2.5.A total of 26 SOC-related QTLs were detected and a single QTL can explain the phenotypic variation?R²?from 3.69%to 18.47%,and the additive effect of a single QTL was between-1.57 and 1.43.These QTLs are mainly distributed on the A01,A03,A05,A06,A09,C01,C03 and C05 chromosomes.The physical position overlap of 2016-qOCA09-1 and 2017-qOCA09-2,2016-qOCA09-2 and BLUP-qOCA09-1,2018-qOCA09-1 and BLUP-qOCA09-2,2016-qOCA05-1 and2016-qOCA05-2,2018-qOCA03-1 and 2018-qOCA03-2,2018-qOCA05-1 and2018-qOCA05-2.These overlapping QTLs are mainly distributed on the A03,A05 and A09 chromosomes,and a single QTL can account for 5.59%-18.47%of phenotypic variation?R²?.The additive effects of these overlapping SNPs are positive,indicating that the synergistic genes affecting seed oil content are mainly from the female parent.2.Genome-wide association study?GWAS?for seed oil content in resequencing natural populationIn this study,a total of 588 B.napus accessions were collected from home and abroad,and all accessions were provided by Chongqing Rapeseed Engineering Technology Research Center and cultivated in the rapeseed planting base of Xiema Town,Beibei District,Chongqing for three consecutive years?2016²018??29°45?39.99"N,106°22?38.47"E,238.57 m?.Combined with 385692valid SNPs of the Brassica napus genome after resequencing screening,the best linear unbiased prediction?BLUP?for the three consecutive years of seed oil content phenotypic data was performed for GWAS.A total of 17 SNPs associated with seed oil content were detected and a single SNP can explain the phenotypic variation from5.46%to 6.68%,and these SNPs were mainly distributed on the A01,A03,C05 and C07 chromosomes.Among them,the A03 chromosome contains the most significant SNPs?11 SNPs?,the C07 chromosome contains 4 significant SNPs,and the A01 and C05 chromosomes contain only one significant SNP.The significant SNPs on the A03and C07 chromosomes are similar of physical position,and the S3₁7975486 on the A03 chromosome can account for the largest phenotypic variation?6.68%?.3.Transcriptome sequencing of high-and low-oil content accessionsSeeds 30 days after flowering are the peak of oil synthesis in Brassica napus at Chongqing.In this study,one low-oil?CQ46,about 33%?and two high-oil?CQ24and CQ52,about 43%?content accessions for 30 days after flowering from seed and silique pericarps on the main inflorescence?30SM and 30SPM,respectively?and on the primary branch?30SB and 30SPB,respectively?were collected for transcriptome sequencing?RNA-Seq?.By conducting venny analysis of CQ24/CQ46 and CQ52/CQ46 in four tissues,we found 1628,2658,2146 and 1493 common DEGs in30SM,30SB,30SPM and 30SPB,respectively.These results provide a wealth of data for subsequent screening for key genes.By conducting GO analysis for common DEGs in 30SM,30SB,30SPM and 30SPB under CQ24/CQ46 and CQ52/CQ46 revealed that the trend of common DEGs enrichment in all four tissues was basically similar.In biological process,common DEGs were mostly enriched in the terms of cellular process,metabolic process and single-organism process.In cellular component,common DEGs were mostly enriched in cell,cell part and organelle.In molecular function,common DEGs were mostly enriched in binding and catalytic activity.In KEGG enrichment analyses,DEGs were significantly enriched in metabolism pathway,especially in carbon metabolism.In this study,we focused on the DEGs in the lipid metabolism pathway and found 21,44,29 and 18 differential lipid metabolism genes in30SM,30SB,30SPM and 30SPB,respectively.4.Screening candidate gene related-SOCBased on the SOC-related candidate genes in confidence interval by QTL mapping and GWAS and combined with DEGs identified by RNA-Seq of extremely high-and low-oil content accessions.We obtained 25 candidate genes related to SOC.Among them,12 genes were up-regulated in high-oil content accessions and 13 were down-regulated.To verify the accuracy of RNA-Seq results,we selected nine genes from DEGs for qRT-PCR validation,and the results showed that the expression trends were similar of these DEGs in qRT-PCR and RNA-Seq results.These results fully confirmed the reliability of RNA-Seq results.5.Screening of the major BnaFAX1 genesIn this study,we identified 21 fatty acid export?FAX?family members in Brassica napus based on homologous genes in Arabidopsis.And evolutionary analysis was conducted of FAX members in Arabidopsis,Brassica rapa,Brassica oleracea and Brassica napus.The gene structure and conserved motifs of FAX members in Brassica napus were compared with FAX members in Arabidopsis.The results showed that among the six BnaFAX1 members,BnaFAX1-1 and BnaFAX1-2 were the closest to AtFAX1 in evolutionary relationship,and the gene structure and conserved motifs were most similar to AtFAX1.By comparing the expression levels of six BnaFAX1 members in different tissues of high-and low-oil content Brassica napus,we found the expression level of BnaFAX1-1 in all tested tissues of high-oil content was higher than that of low-oil content accession.The expression level of BnaFAX1-2 in most of tested tissues of high-oil content was higher than that of low-oil content accession,BnaFAX1-3 was not expressed in all tested tissues,and the expression level of BnaFAX1-4 in all tested tissues was relatively low.Although the expression levels of BnaFAX1-5 and BnaFAX1-6 in each tested tissue were relatively high,their expression levels in low-oil content accession were basically higher than those in high-oil content accession.Therefore,we speculate that BnaFAX1-1 and BnaFAX1-2 may play a major role in the oil accumulation of high-oil content in Brassica napus.Overexpression of these two genes may contribute to increase seed oil content in Brassica napus.In our previous study,BnaFAX1-1?14454789-14456198?was located at upstream 91 kb of the biological yield significant SNP?Bn-A07-p12412116?at14546056.Therefore,in this study,two genes?BnaFAX1-1 and BnaFAX1-2?were selected for further transgenic study.6.Effects of heterologous overexpression of BnaFAX1-1 and BnaFAX1-2 genes in Arabidopsis on plant growth and developmentBy comparing the growth phenotypes of wild-type and heterologous overexpression BnaFAX1-1 and BnaFAX1-2 Arabidopsis plants,we found the heterologous overexpressing BnaFAX1-1 and BnaFAX1-2 lines grew more vigorously.Detailed analysis of different tissues and organs about transgenic and WT plants grown for 49 days was performed,and we found stem fresh weigh,stem diameter,plant height,rosette fresh and dry weight,fresh and dry weight of biological yield above ground?BY?in overexpressing BnaFAX1-1 and BnaFAX1-2 lines were significantly higher than that of WT plants.After seeds matured,length of siliques,number of siliques per plant,number of seeds per silique,weight of per 1000 seeds and weight of seeds per plant were counted.The results showed that number of siliques per plant and weight of seeds per plant in BnaFAX1-1 overexpressing lines were significantly higher than that of WT plants,while length of siliques,number of seeds per silique and weight of per 1000 seeds are similar between BnaFAX1-1 overexpressing and WT plants.However,except for weight of per 1000 seeds,the length of siliques,number of siliques per plant,number of seeds per silique and weight of seeds per plant in BnaFAX1-2 overexpressing lines were significantly higher than that of WT plants.The above results indicated that heterologous overexpression of BnaFAX1-1 and BnaFAX1-2 in Arabidopsis can not only promote plant growth and development,but also increase seed yield per plant.Besides,the total lipid content in mature seeds was determined.The results showed that the total lipid content of BnaFAX1-1overexpressing seeds was significantly higher than that of WT,while BnaFAX1-2overexpressing lines did not differ significantly from WT.7.Effects of overexpression of BnaFAX1-1 and BnaFAX1-2 genes in Brassica napus on plant growth and developmentWe investigated the phenotypes of flowering plants?grown 175-day plants?.Compared with WT,overexpressing BnaFAX1-1 and BnaFAX1-2 lines showed stronger growth,higher plant height,more and larger leaves.To more accurately understand the growth differences between transgenic and WT plants,we randomly selected 10individuals between each line for phenotypic determination.The results showed that the plant height,the number of leaves on the main stem,leaf length,leaf width,leaf area,diameter of the upper,middle and lower stem of overexpressing BnaFAX1-1 and BnaFAX1-2 lines were significantly higher than that of WT plants?The leaves and stems at the same location of per plant were selected for measurement?,while the chlorophyll content and photosynthetic rate were not significantly different from WT.After seeds matured,the phenotypes of BnaFAX1-1 and BnaFAX1-2 overexpressing Brassica napus lines and WT plants were observed and measured.The results showed that the plant height,effective branch number per plant,silique number per main inflorescence,silique number per plant,silique length,seed number per silique,seed yield per plant and biomass yield above ground of BnaFAX1-1 and BnaFAX1-2overexpressing lines were significantly higher than WT plants,while there was no difference in the length of the main inflorescence.There was no significant difference in weight per 1000 seeds of BnaFAX1-1 transgenic seeds compared to WT,while weight per 1000 seeds of BnaFAX1-2 transgenic seeds was significantly reduced.These results indicated that promoting the expression of BnaFAX1-1 and BnaFAX1-2genes in Brassica napus can effectively promote the plant growth and development and effectively increase the seed yield of Brassica napus.8.Effects of BnaFAX1-1 and BnaFAX1-2 genes overexpression on seed oil content and fatty acid composition in Brassica napusThe mature seed oil and protein content of BnaFAX1-1 and BnaFAX1-2overexpressing lines and WT plants were determined.The results showed that the seed oil content in the BnaFAX1-1 and BnaFAX1-2 transgenic mature seeds was significantly higher than WT,and there was no significant difference in seed protein content.Further analysis of the fatty acid composition in mature seeds showed that the oleic acid?C18:1?content in the BnaFAX1-1 and BnaFAX1-2 transgenic lines was significantly increased compared to WT plants,while arachidic acid?C20:0?and erucic acid?C22:1?content was significantly reduced.These results fully demonstrated that BnaFAX1-1 and BnaFAX1-2 overexpression in Brassica napus can simultaneously increase seed oil content and quality.Taken together,the results indicated that BnaFAX1-1 Simultaneously increases the seed oil content and seed yield in Brassica napus,while its homologous gene in Arabidopsis does not have a similar phenotype.This indicated that BnaFAX1-1 has a more important role in Brassica napus.